This invention relates generally to the production of hybridoma proteins and more specifically to the production of hybridoma antibodies.
It has been postulated that if one could isolate one cell making a single specific antibody and grow it in culture, the cell's progeny or clone would be a source of large amounts of identical antibody against a single antigenic determinant-a monoclonal antibody. Unfortunately, antibody-secreting cells cannot be maintained in a culture medium.
There are malignant tumors of the immune system called myelomas, however, whose rapidly proliferating cells can produce large amounts of abnormal immunoglobulins called myeloma proteins in a culture medium. In more basic terms, a myeloma is an old tumor which is no longer capable of producing antibodies (a plasmocytoma). In 1975, investigators learned how to fuse mouse myeloma cells with lymphocytes from the spleen of mice immunized with a particular antigen. The resulting hybrid-myeloma, or "hybridoma", cells express both the lymphocyte's property of specific-antibody production and the immortal character of the myeloma cells.
By following the fusing or splicing technique discussed above which is described in articles entitled "Antibody Reagents Revolutionizing Immunology" by Jeffrey L. Fox, Jan. 1, 1979, C & EN, and "Monoclonal Antibodies" by Yelton and Scharff, American Scientist, Vol. 168, pp. 510-516, cells that secrete antibodies can be made immortal by fusing them with tumor cells and cloning the hybrids. Each clone is a long-term source of substantial quantities of a single highly specific antibody. Highly specific monoclonal antibodies produced by this general method have proved to be a remarkably versatile tool in many areas of biological research and clinical medicine. It is these hybridomas which produce antibodies or "hybridoma antibodies" as they can be called toward which the present invention is directed.
Once an antibody producing clone is produced from a hybridoma, cloned cells can be utilized to produce antibodies in two ways. One method is to inject the cloned cells into the belly of a mouse. While in the belly of the mouse, the antibody producing clone proliferates and the antibody it makes becomes concentrated by the mouse in the fluid of the belly (ascitic fluid) and in the blood. The antibody is harvested by tapping the fluid from the belly atraumatically with a needle and syringe. A major disadvantage, however, of raising the hybridoma antibody in vivo is that in the process, the hybridoma product becomes mixed with all of the other immunoglobulins present in the mouse and is no longer monoclonal.
Of course the alternative to any in vivo procedure like the one discussed above is an in vitro procedure. The problem with producing antibodies from hybridoma in culture, however, results from the fact that the hybridoma releases the antibody in the culture medium to produce a very dilute antibody containing liquid. Indeed the amount of antibody in a tissue culture supernatant is typically in the order of about 10 micrograms per milliliter. As can be appreciated, recovery of an antibody from such dilute supernatant greatly increases recovery costs.
However, an in vitro procedure has at least one distinct advantage over an in vivo procedure. The marked advantage of the in vitro procedure is that the culture conditions can be easily arranged so that the species of immunoglobulin being produced in the culture (the hybridoma product) is different from that of any other which may be contaminating the culture medium. For example, bovine serum normally has to be added to the culture system and thus bovine immunoglobulins will be mixed with the hybridoma product (normally mouse or rat).
The important point is that in an in vitro production method, conditions can be easily adjusted so that the hybridoma product being grown is unique with regard to its derivative species and that is not the case with the fluid recovered from the belly of an animal.
The major problem to be dealt with, in raising hybridoma antibodies or other proteins in vitro is to devise a method of purifying and concentrating the desired monoclonal antibody without co-purifying the chemically similar immunoglobins derived from the serum supplement to the tissue culture medium. Of course it would be highly desirable to have a reagent which is specific for the protein to be recovered (mouse immunoglobulin for example).
In short, hybridoma antibodies and other proteins produced in tissue culture occur at very low concentration and thus are difficult to purify in good yield at reasonable cost. On the other hand, proteins such as antibodies produced in vivo are contained in a fluid with other proteins which present purification problems. Both problems would be reduced if one had available large quantities of a reagent that binds mouse immunoglobulins or other proteins specifically.